Detection Method

ABSTRACT

The described systematics for detecting a substance or substances in a sample or in a matrix of samples, by way of the combination of detection methods on the one hand based on direct detection with integrated optical (bio)chemo-sensitive waveguide grating structures and on the other hand based on a mass-spectrometric detection effected by way of a desorption process, permit an increase of the detection security and/or detection sensitivity.

[0001] Optical chemo- and biosensors based on integrated optical(bio)chemo-sensitive or (bio)chemo-functional waveguide gratingstructures permit the marking-free detection of (bio)molecularinteractions in real time and are described in the literature (see e.g.U.S. Pat. No. 4,815,843, U.S. Pat. No. 5,071,248. However markingdetections are also possible (see e.g. WO 99/13320).

[0002] The method according to the invention not only represents adetection method, but also a separation technology since the substanceto be tested or the substances to be tested, of the sample or of thematrix of samples, are separated in that the substance(s) binds(bind)onto the (bio)chemo-sensitive layer(s) located on the sensor chip.

[0003] A marking-free detection is effected in a first step, as isdescribed in U.S. Pat. No. 4,815,843, U.S. Pat. No. 5,071,248, U.S. Pat.No. 5,738,825, U.S. Pat. No. 5,479,260 and EP 0,482,377. Subsequentlythe substance (or parts (fragments) thereof) to be detected and bindingonto the (bio)chemo-sensitive layer on the sensor chip is analysed moreexactly in a mass spectrometer with a desorption step (and ionisationstep).

[0004] The present invention describes detection systematics in whichintegrated optical chemo- and biosensorics (with or without markingtechnology (index marker, fluorescence marker, luminescence marker,phosphorescence marker, enzyme marker)) are combined with massspectroscopy with a desorption step (and ionisation step). Thedesorption step releases the molecules from the surface of the sensorchip. The mass spectrometer measures the masses and/or the degree ofionisation of the molecules (atoms, ions, biomolecules, fragments etc.).The present invention thus creates detection systematics which furtherincreases the detection sensitivity as well as the security ofdetection.

[0005] Integrated optical chemo- and biosensors are above all to beunderstood as those integrated optical sensor chips, which are based on(bio)chemo-sensitive or (bio)chemo-functional waveguide gratingstructures. A waveguide grating structure consists of at least onewaveguide grating structure unit (with or without reference waveguidegrating structure unit) or of at least one sensor location (with orwithout reference sensor location). A waveguide grating structurecomprises at least one grating location). A waveguide grating structureunit comprises at least one grating, but may however also have at leastone in-coupling grating and one out-coupling grating. The in-couplinggrating and the out-coupling grating may have the same or differentgrating periods. A waveguide grating structure may also consist of onlyone large (uni-diffractive or multi-diffractive) grating. A waveguidegrating structure may contain several sensor pads (two, three, fouretc.) lying next to one another and/or in one another and/or over oneanother, in order e.g. to excite a TE-wave (preferably in the base mode)in the forwards and rearwards direction and a TM-wave (preferably in thebase mode) in the forwards and rearwards direction.

[0006] Chiefly highly-diffractive materials are considered with regardto waveguiding films, such as TiO₂, Ta₂O₅, ZrO₂, HfO₂, Si₃O₄ etc. Thewaveguide (the waveguide structure) is mainly a monomode structure, thuscarries only the base modes TE0 (TE=transversal electrical, mode numberm=0) and TM0 (TM=transversal magnetic, mode number m=0). A waveguidestructure may contain several layers, wherein preferably of these atleast one layer is highly diffractive. The waveguide (the waveguidestructure) may also be light-absorbing. At the same time the absorbingmaterial may be embedded into a layer or into the substrate, or also anabsorbing layer or an absorbing substrate may be present. An absorbinglayer may e.g. be a metal layer (chromium, aluminium, nickel, gold,silver etc).

[0007] Apart from (bio)molecular binding partners (such as e.g.antibodies, antigenes, receptors, peptides, phages, “single-stranded”DNA(RNA)-sections, genes, gene sections, targets, proteins, bindingproteins, enzymes, inhibitors, nucleic acids, nucleotides,oligonucleotides, SNP, allergens, pathogens, carbohydrates, metabolites,hormones, active ingredients, molecules with low molecular weight,lipids, signal substances etc.) one may also apply “molecular imprintedpolymers” (such as plastic antibodies, plastic antigenes etc.) or(living) cells as (bio)chemo-functional layers. The binding procedures(or the (bio)chemical reactions) may also be effected here at thesurface, in the volume or on the surface as well as in the volume of the(bio)chemo-functional layer.

[0008] The (bio)chemo-functional layer may at the same time lie on a(uni-diffractive or multi-diffractive) waveguide grating or between two(uni-diffractive or multi-diffractive) waveguide gratings (of the sameor different grating period and/or modulation). In the later case thetwo gratings are construed as belonging to one waveguide gratingstructure unit. In the case of scatter light measurements orflourescence, luminescence or phosphorescence measurements, the(bio)chemo-functional layer may also be located next to the waveguidegrating. The (bio)chemo-functional layers may also cover a largewaveguide grating in an array-like (matrix-like or circular) manner,without the (bio)chemo-functional layers overlapping. The(bio)chemo-functional layers (signal layer and/or reference layers)define the sensor locations. A passivation material which whereappropriate suppresses non-specific binding (NSB) may (need not) belocated between the (bio)chemo-sensitive layers.

[0009] A (removable) sample accommodation device (e.g. (removable)cuvette, a (removable) well or a (removable) through-flow or capillarycuvette) or an array of sample accommodation devices may be locatedabove the (bio)chemo-sensitive waveguide grating structure. The well ismostly a component of a well plate. The sample accommodating device mayhowever also be incorporated into the waveguide grating structure.Photolithography, laser ablation, (hot) embossing technology or plastic(hot) embossing technology or injection moulding technology are suitableas manufacturing methods. The recesses in the substrate act as wells. A(channel-like) recess with a cover plate (provided with supply andremoval bores) may act as a through-flow cuvette, a (channel-like)recess with a (part) covering may act as a capillary cuvette. Thewaveguide grating structure need not necessarily be provided with asample accommodating device. For example the samples may be depositedwith a pipetting robot in the form of drops. During measurement theinjection needles or pipette tips of the pipetting robot may (or maynot) remain in contact with the sample droplet deposited on the sensorchip.

[0010] The inner wall of a through-flow channel of a lab-on-chip may beprovided with a (bio)chemo-sensitive waveguide grating structure. If thecover plate of the through-flow channel is removed, then the(bio)molecules accumulated on the (bio)chemo-sensitive waveguide gratingstructure may be brought to desorb into the upper half space by way of adesorption process (e.g. LDI-process or MALDI-process). Preferably nosample fluid is located in the through-flow channel during thedesorption procedure. However one may also operate without the removalof the cover plate. The MALDI-matrix may e.g. be supplied via a sampleloop. The lab-on-chip or the cover plate is transparent to the laserradiation which triggers the desorption. The desorbed (bio)moleculesmigrate along the through-flow channel and at the end get into thevacuum of the mass spectrometer. If working in the liquid phase, theoutlet of the through-flow channel may e.g. be connected to anelectrospray-ionization part of a mass spectrometer (quadrupole-,tandem-, time-of-flight mass spectrometer etc.).

[0011] It is not good to add plastic parts to the vaccum of the massspectrometer since plastic parts desorb material into the vaccum. Forthis reason it is also advantageous to use sensor chips on glasssubstrates. The photo-lithographic manufacture of gratings in glass(with wet or dry etching) or the manufacture of the gratings with glassembossing technology is known from the literature. Gratings in plasticsubstrates may e.g. be manufactured with (hot) embossing technology orinjection moulding technology (with or without compression step(s)).

[0012] The direct detection of a binding in the case that the(bio)chemo-sensitive layer is located on the grating is effected e.g. byway of an in-coupling angle measurement or an out-coupling anglemeasurement or a wavelength measurement (see U.S. Pat. No. 4,815,843) oran interferometric measurement (see U.S. Pat. No. 5,479,260), and in thecase that the (bio)chemo-sensitive layer is located between twowaveguide gratings (of the same or different grating period) is effectedby way of an interferometric measurement (see Biosensors &Bioelectronics 6 (1991), 215-225, European Patent 0 226 604 B1).Interferometric measurements may for example be based on theMach-Zehnder principle, wherein both light paths may be guided separatedfrom one another via the in-coupling grating and out-coupling grating.One light path sees the (bio)chemo-functional layer, the other lightpath sees another or inert or even no (bio)chemo-functional layer. Suchmeasuring technology has been described by R. G. Heideman et al.,“Development of an Optical Waveguide Interferometric Immunosensor”Proceedings Eurosensors 4, Karlsruhe, 1990. In our case a transparentsubstrate is preferred in order to permit light incidence from thesubstrate side.

[0013] Another measuring technology is based on the measurement ofemission light (fluorescence, luminescence, phosphorescence light) onwaveguide (grating) structures in combination with a direct measurement.With this, a layer of the waveguide structure (consisting of one or morelayers) and/or a layer between the waveguiding film and the substrateand/or a layer between the waveguiding film and cover (or(bio)chemo-functional layer) and/or a layer on the underside of thesubstrate and/or the substrate itself is light-emitting (fluorescence,luminescence, phosphorescence light) on excitation with light (with awide and/or narrow excitation spectrum). This layer may e.g. be apolymer layer (or a solid-state-like layer or a glass-like layer) with a(high) intrinsic fluorescence or with embedded emission light molecules(fluorescence, luminescence, phosphorescence molecules). The emissionlight wavelength is different to the excitation wavelength. The methodsystematics are based on the incident angle scanning mode or on awavelength scanning mode (with a matchable light source), wherein in thebeam path between the sensor chip and the detector there is located awavelength filter (blocking the excitation light, transparent to theemission light). A beam splitter may also be located between the sensorchip and the detector, wherein then the wavelength filter is preferablyin the beam path between the beam splitter and detector. The excitationlight may e.g. be incident onto the sensor chip ((bio)chemo-functionalwaveguide grating structure) via the beam splitter. The excitation lightmay also be incident obliquely from the substrate side or obliquely fromthe cover side onto the sensor chip, wherein the beam splitter may ormay not be present. The emission light may also (preferably) be measuredin a direction which does not correspond to the reflection direction ofthe excitation light beam. The incident light beam on fulfilling thein-coupling equation produces a guided light wave, but may also exciteemission light which by way of the led mode is directly or indirectly(resonance-like) intensified and/or shifted with respect to centre ofintensity. The (radiated and/or out-coupled) emission light of theemission layer is imaged onto the detector with a lens (lens system).The out-coupled light of the excitation wave may or may not be incidentonto the imaging lens. The out-coupled emission light may or may not beincident onto the imaging lens. The measuring systematics represents acombination of direct measurement with fluorescence (luminescence,phosphorescence) measurement. Since with the wavelength scanning modethe (excitation) wavelength is shifted, the excitation spectrum must besufficiently broad. Light-emitting mode-beating patterns (between the TEmode and TM mode) and light-emitting interferometric patterns with andwithout (uni-diffractive or multi-diffractive) gratings with scanningoperation (incident angle scanning mode or wavelength scanning mode) orwithout scanning operation (excitation of the modes TE and/or TM withplanar or slightly focussed waves) may be measured with the applicationof polariser (e.g. 45° polariser) located between the sensor chip andthe detector in the case of interference of TE-light and TM-light, andwith the application of the mentioned wavelength filter at the point intime of mode excitation, wherein the modes of the excitation wavelenegthare produced via grating in-coupling. A binding reaction (or massaccummulation) or chemical reaction (change of the complex refractiveindex) on the (bio)chemo-functional layer which is located on and/ornext to the grating, changes the period of the interference pattern.

[0014] In place of removing the well plate, the well plate may also bebrought into contact with the vacuum in a manner such that only thesensor locations come into contact with the vacuum, but not the sampleplate. This is effected by way of the fact that hollow cylinders areintroduced into the wells which amongst one another are again connectedto one another in a vacuum-tight manner. The above complicated designbecomes invalid if however the wells are introduced into the waveguidegrating.

[0015] There are however also sample plates of glass or plastic whichlikewise are subjected to the vacuum.

[0016] One advantageous embodiment of an integrated optical (IO) sensorchip or IO-sensor chip plate is a micro-plate with e.g. 24, 48, 96, 384,1536 wells or a sensor chip array (e.g. micro-array) with any number ofsensor locations. Microplates are described in U.S. Pat. No. 5,738,825and WO 99/13320. In WO 99/13320 it is further described howtemperature-compensated marking-free detection technology functions.With the micro-array the (bio)chemo-sensitive layers are advantageouslydeposited with a spotter or a contact-printing robot in a matrix-like(or also circular) manner. A micro-array or also generally a waveguidegrating structure—may comprise a (location-dependent or alsonon-location dependent) absorbing or also non-absorbing waveguide. Amicro-array— or also generally a waveguide grating structure—may have alarge extended (uni-diffractive or multi-diffractive) grating with anarray of (bio)chemo-functional layers or also consist of an array of(bio)chemo-functional waveguide grating structure units. A waveguidegrating structure unit in each case is at least partly covered by a(bio)chemo-sensitive layer. The microarray may (need not) be providedwith a (removable) fluid receptacle (cuvette, well, through-flow cell,capillary cuvette etc.).

[0017] The (bio)chemo-sensitive ((bio)chemo-functional) layers (signallayers and/or reference layers) are preferably deposited with a spotteror contact-printing robot or a liquid handler, wherein a linker-layer oralso a (absorbing or non-absorbing) distance layer may be locatedbetween the (bio)chemo-functional layer and the waveguide gratingstructure. Micro-arrays are e.g. applied in genomics or proteomics. Ingenomics the (bio)chemo-functional layers are e.g. gene sections,nucleic acids, single stranded DNA (RNA), single nucleotide polymorphism(SNP) etc.. In proteomics the (bio)chemo-functional layers are e.g.proteins, phages etc..

[0018] The particular advantage of grating-based integrated opticalchemo- and biosensors is their ability to be automised, since eachsensor location is simply addresseable via diffraction. Sensor locationsmay be illuminated after one another or simultaneously. Simultaneousillumination of the sensor locations may be effected with several beamsor with a diverged beam. The beam may contain a wavelength or several(discrete or continuous) wavelengths.

[0019] A MALDI step (matrix assisted laser desorption ionisation) mayserve as a desorption/ionisation step for the mass spectrometer. Thisdesorption process produces molecules in the ionised condition. TheMALDI matrix is not compellingly required in some applications. A laserdesorption and ionisation (LDI) may also take place without a MALDImatrix or with a different desorption and ionisation source. Whereappropriate one adds yet a separate ionisation step.

[0020] Various MALDI matrixes are used according to the analyte. TheMALDI matrices are described in the literature. Typical MALDI matricesare derivatives of the cinnamic acid, alpha-cyano-4-hydroxycinnamicacid, gentisic acid, dithranol, sinapinic acid etc.

[0021] The desorption process may however also be triggered by an ionsource (or ion beam), an atomic source (or atomic beam), an electronsource (or electron beam), an X-ray source (or x-ray beam) etc..

[0022] A TOF (time of flight) mass spectrometer may e.g. be applied.Other mass spectrometers are magnetic sector mass spectrometers, iontrap mass spectrometers, quadrupole mass spectrometers, tandem massspectrometers, dual quadrupole mass spectrometers, triple quadrupolemass spectrometers, Fourier transform ion cyclotron resonance massspectrometers etc..

[0023] The (bio)chemo-sensitive waveguide grating structure may beapplied in a pre-chamber of the mass spectrometer. This pre-chamber isthen evacuated. The chamber of the mass spectrometer remains under avacuum. If the pre-chamber is evacuated then the sluice between thepre-chamber and the chamber may be opened. However the(bio)chemo-sensitive waveguide grating structure may be applied into thechamber of the mass spectrometer and the chamber subsequently beevacuated.

[0024] The mass spectrometer measures e.g. in the mass spectrum theratio of m/z of mass to charge. The peak height of a peak in the massspectrum is a measure of the quantity of analytes which are ionised anddetected by the mass spectrometer. The sensor chip (e.g. single-channelchip, multi-channel chip, microplate, microarray, lab-on-chip, disc chipetc) is applied into the measuring device, then a vacuum is produced andsubsequently the desorption process is activated. The desorbed moleculesor ions are analysed in the mass spectrometer.

[0025] Liquid samples may be ionised via an electro-spray ionisationstep (ESI) and subsequently led to the mass spectrometer.

[0026] The MALDI matrix may be deposited onto the sensor chip with a(bio)chemo-functional layer and possibly a substance to be detectedwhich is bonded thereon, in the liquid phase as well as in the gasphase.

[0027] The MALDI matrix is shot with a pulsed or non-pulsed laser(wavelength region: X-ray, gamma, UV, VIS, IR). Pulsed lasers are e.g. anitrogen laser or a (Q-switched) Nd-YAG laser where appropriate with afrequency doubling, or frequency tripling or a frequencyquadruplication, or an Erbium-YAG laser.

[0028] The laser beam or the sensor chip (the waveguide gratingstructure) may be displaced for traversing to a measuring location.

[0029] The laser beam which is responsible for the desorption mayimpinge the (bio)chemo-functional layer (with possibly bonded substance)from the substrate side as well as from the cover side. The incidencefrom the substrate side requires transparency of the substrate withrespect to the laser wavelength. However the laser beam responsible forthe desorption may be coupled into the waveguide structure from thecover side or from the substrate side via a waveguide grating. Withhighly diffractive waveguiding films (or with a large difference in therefractive index between the substrate and the waveguiding film), as isknown, the electromagnetic field strength of the evanescent wavereaching into the (bio)chemo-functional layer is particularly high. Inthis case the evanescent wave at least also takes part in thedesorption.

[0030] With the (pulsed or non-pulsed) laser beam (with reduced power)responsible for the desorption it is also possible to make a directdetection (in real time or as an end-point measurement (with regard tothe initial condition)) or to make a direct detection with a secondcontrol laser (e.g. HeNe laser or laser diode) or to follow thedesorption procedure using a grating coupling principle (e.g. incidentangle scanning mode, out-coupling angle scanning mode, wavelengthscanning mode) or an interferometric principle. For this one requiresoptics and detectors and measuring means (for the absolute measurement),as e.g. are described in a second patent application with the samepriority by the company Artificial Sensing Instruments ASI AG. Thedetectors are preferably not located in a vacuum, but may also belocated in a vacuum.

[0031] The MALDI matrix absorbs the laser light and with this triggersthe desorption. If marking substances are present on the(bio)chemo-functional layer or on the accumulated substances to bedetected, then the molecular weight (and the ionisation) of the markingsubstance or the desorbed marking substance fragments in the massspectrum must be taken into account.

[0032] The advantage of the detection systematics according to theinvention is that with the integrated optical chemo- and biosensoricsthere may be effected a rapid direct detection at several sensorlocations (on a one-dimensional or two-dimensional array of sensorlocations) and subsequently in a vacuum at selected sensor locationsthere may be effected a more time-consuming, but more accuratemass-spectroscopic analysis on the bonded substance (or on partsthereof).

1. A method for the detection of a substance or of substances in asample or in a matrix of samples, characterised in that (a) thesubstance to be detected or the substances to be detected is separatedfrom the sample or matrix of samples with an integrated optical(bio)chemo-sensitive sensor-chip plate based on at least one(bio)chemo-sensitive waveguide grating structure unit or(bio)chemo-sensitive sensor location, or with an integrated optical(bio)chemo-sensitive micro-array based on at least one(bio)chemo-sensitive waveguide grating structure unit or(bio)chemo-sensitive sensor location, or with an integrated optical(bio)chemo-sensitive lab-on-chip based on at least one(bio)chemo-sensitive waveguide grating structure unit or(bio)chemo-sensitive sensor location, (b) a detection of the substanceor the substances is effected during or after the separation with thehelp of a light wave which has an evanescent field and which at leastpartly is guided in the waveguide structure (c) a detection of thesubstance or the substances or of substance parts on selected(bio)chemo-sensitive waveguide grating structure units or on selected(bio)chemo-sensitive sensor locations is effected by desorption andionisation of the substance or the substances or of substance parts withthe help of a mass spectrometer.
 2. A method according to claim 1,characterised in that the (bio)chemo-sensitive layer at least partlycovers the waveguide grating structure unit or the sensor location of awaveguide grating.
 3. A method according to claim 1, characterised inthat the (bio)chemo-sensitive layer covers a diffracting part of awaveguide grating structure unit.
 4. A method according to claim 1,characterised in that the (bio)chemo-sensitive layer covers anon-diffracting part of a waveguide grating structure unit.
 5. A methodaccording to claim 1, characterised in that the waveguide grating or thewaveguide grating structure unit contains a uni-diffractive waveguidegrating.
 6. A method according to claim 1, characterised in that thewaveguide grating or the waveguide grating structure unit contains amulti-diffractive waveguide grating.
 7. A method according to claim 1,characterised in that a waveguide grating structure unit consists of twowaveguide gratings lying next to one another, both waveguide gratingsbelong to the same waveguide grating structure unit and thenon-diffracting distance between the two waveguide gratings is part ofthe waveguide grating structure unit.
 8. A method according to claim 1,characterised in that the waveguide grating structure unit consists oftwo part-waveguide grating structures lying next to one another and thenon-diffracting distance between the two part-waveguide gratingstructures is part of the waveguide grating structure.
 9. A methodaccording to claim 1, characterised in that the waveguide grating or thewaveguide grating structure contains a one-dimensional ortwo-dimensional array of sensor locations.
 10. A method according toclaim 1, characterised in that the sensor chip plate is a micro-plate.11. A method according to claim 1, characterised in that the sensor chipplate or the micro-array or the lab-on-chip comprises a glass substrate.12. A method according to claim 1, characterised in that the sensor chipplate or the micro-array or the lab-on-chip comprises a polymersubstrate.
 13. A method according to claim 1, characterised in that thesensor chip plate or the micro-array or the lab-on-chip comprises aremovable sample accommodating device or sample holder device.
 14. Amethod according to claim 1, characterised in that the sensor chip plateor the micro-array or the lab-on-chip comprises a sample accommodatingdevice or a sample holding device of glass.
 15. A method according toclaim 1, characterised in that the sensor chip plate or the micro-arrayor the lab-on-chip comprises a sample accommodating device or a sampleholder device of polymer.
 16. A method according to claim 1,characterised in that the desorption and ionisation is effected byshooting with a pulsed laser.
 17. A method according to claim 1,characterised in that a (bio)chemo-sensitive waveguide grating structureunit with accumulated analyte or a (bio)chemo-sensitive sensor locationof a waveguide grating with accumulated analyte is provided with a MALDImatrix.
 18. A method according to claim 1, characterised in that thedesorption and ionisation is effected via a MALDI step.
 19. A methodaccording to claim 1, characterised in that the mass spectrometer is atime-of flight (TOF) mass spectrometer or a quadrupole massspectrometer.